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Yu-Qian Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
University of Chinese Academy of Sciences, Beijing, China

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Aalia Batool State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
University of Chinese Academy of Sciences, Beijing, China

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Su-Ren Chen State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

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Yi-Xun Liu State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

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Reduced contractility of the testicular peritubular myoid (PTM) cells may contribute to human male subfertility or infertility. Transcription factor GATA4 in Sertoli and Leydig cells is essential for murine spermatogenesis, but limited attention has been paid to the potential role of GATA4 in PTM cells. In primary cultures of mouse PTM cells, siRNA knockdown of GATA4 increased the contractile activity, while GATA4 overexpression significantly attenuated the contractility of PTM cells using a collagen gel contraction assay. Using RNA sequencing and qRT-PCR, we identified a set of genes that exhibited opposite expressional alternation between Gata4 siRNA vs nontargeting siRNA-treated PTM cells and Gata4 adenovirus vs control adenovirus-treated PTM cells. Notably, ion channels, smooth muscle function, cytokines and chemokines, cytoskeleton, adhesion and extracellular matrix were the top four enriched pathways, as revealed by cluster analysis. Natriuretic peptide type B (NPPB) content was significantly upregulated by GATA4 overexpression in both PTM cells and their culture supernatant. More importantly, the addition of 100 μM NPPB could abolish the promoting effect of Gata4 silencing on PTM cell contraction. Taken together, we suggest that the inhibitory action of GATA4 on PTM cell contraction is mediated at least partly by regulating genes belonging to smooth muscle contraction pathway (e.g. Nppb).

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Yu Chen School of BioSciences, The University of Melbourne, Victoria, Australia

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Jaiden Lay School of BioSciences, The University of Melbourne, Victoria, Australia

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Geoffrey Shaw School of BioSciences, The University of Melbourne, Victoria, Australia

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Gerard A Tarulli School of BioSciences, The University of Melbourne, Victoria, Australia

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Marilyn B Renfree School of BioSciences, The University of Melbourne, Victoria, Australia

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In brief

Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation.

Abstract

The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20–40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17β-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17β-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40–50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.

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Yu Chen School of BioSciences, The University of Melbourne, Victoria, Australia

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Hongshi Yu School of BioSciences, The University of Melbourne, Victoria, Australia

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Andrew J Pask School of BioSciences, The University of Melbourne, Victoria, Australia

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Asao Fujiyama Advanced Genomics Center, National Institute of Genetics, Mishima, Shizuoka, Japan

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Yutaka Suzuki Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan

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Sumio Sugano Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan

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Geoff Shaw School of BioSciences, The University of Melbourne, Victoria, Australia

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Marilyn B Renfree School of BioSciences, The University of Melbourne, Victoria, Australia

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The development of the mammalian phallus involves hormone-dependent mesenchymal–epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

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Min Yu Obstetrics & Gynecology Hospital of Fudan University, Shanghai JIAI Genetics & IVF Institute, Shanghai, China

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Xiandong Peng Obstetrics & Gynecology Hospital of Fudan University, Shanghai JIAI Genetics & IVF Institute, Shanghai, China

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He Li Obstetrics & Gynecology Hospital of Fudan University, Shanghai JIAI Genetics & IVF Institute, Shanghai, China

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Yining Xu Obstetrics & Gynecology Hospital of Fudan University, Shanghai JIAI Genetics & IVF Institute, Shanghai, China

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Xiaoxi Sun Obstetrics & Gynecology Hospital of Fudan University, Shanghai JIAI Genetics & IVF Institute, Shanghai, China
Shanghai Key Laboratory of Female Reproductive and Endocrine-Related Diseases, Shanghai, China

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Jiazhou Chen Obstetrics & Gynecology Hospital of Fudan University, Shanghai JIAI Genetics & IVF Institute, Shanghai, China

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Embryo implantation, a critical step during the mammalian reproductive process, requires normal developing blastocysts and a receptive endometrium. Endometriosis, a common pathologically benign gynecological condition, is associated with decreased fertility and reduced endometrial receptivity. The oncoprotein, Gankyrin, has been associated with endometriosis and endometrial cancer. Here, we examined the role of Gankyrin during the process of embryo implantation and found that Gankyrin expression levels were significantly increased during the mid-secretory phase, but unaffected during the proliferative phase in the human endometrium. Using an in vitro cell adhesion assay to examine the cell adhesion rate of BeWo trophoblast spheroids to Gankyrin knockdown or overexpressing human endometrial carcinoma RL95-2 cells, we demonstrated that the adhesion rate was significantly reduced in Gankyrin-knockdown RL95-2 cells, while overexpression of Gankyrin promoted cell adhesion. Furthermore, we found that the downregulation of Gankyrin inhibited STAT3 activation and subsequent matrix metalloproteinase 2 (MMP2) expression, while overexpression led to STAT3 activation and MMP2 expression. In vivo, we found that Gankyrin expression was increased in the endometrium after conception but decreased with the prolongation of gestation time in female mice. siRNA-mediated knockdown of Gankyrin in the uterine horn led to a significant reduction in the number of implanted embryos 9 days post-gestation, which was associated with a decrease in p-STAT3 expression and MMP2 transcription. Taken together, our findings indicate that Gankryin has a potential role in embryo implantation via STAT3 activation.

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Liuhong Yang Key Laboratory for Regenerative Medicine (JNU-CUHK), Department of Developmental and Regenerative Biology, Ministry of Education,

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Lei Chen Department of Anesthesiology, The First Affiliated Hospital, Jinan University, Guangzhou, People’s Republic of China

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Xiaosheng Lu Key Laboratory for Regenerative Medicine (JNU-CUHK), Department of Developmental and Regenerative Biology, Ministry of Education,

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Anni Tan Key Laboratory for Regenerative Medicine (JNU-CUHK), Department of Developmental and Regenerative Biology, Ministry of Education,

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Yao Chen Key Laboratory for Regenerative Medicine (JNU-CUHK), Department of Developmental and Regenerative Biology, Ministry of Education,

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Yalan Li Department of Anesthesiology, The First Affiliated Hospital, Jinan University, Guangzhou, People’s Republic of China

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Xuemei Peng Department of Anesthesiology, The First Affiliated Hospital, Jinan University, Guangzhou, People’s Republic of China

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Shaochun Yuan State Key Laboratory of Biocontrol, Department of Biochemistry, School of Life Sciences, Sun Yat-Sen University, Guangzhou, People’s Republic of China

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Dongqing Cai Key Laboratory for Regenerative Medicine (JNU-CUHK), Department of Developmental and Regenerative Biology, Ministry of Education,

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Yanhong Yu Key Laboratory for Regenerative Medicine (JNU-CUHK), Department of Developmental and Regenerative Biology, Ministry of Education,

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Peri-ovarian adipose tissue (POAT) is a kind of intra-abdominal white adipose tissue that is present surrounding the ovaries in rodents. Recent studies demonstrated that POAT-deficient mice displayed a phenotype of delayed antral follicular development, for which decreases in serum estrogen, serum FSH and FSHR levels were responsible. However, folliculogenesis is regulated by endocrine signals and also modulated by a number of locally produced intraovarian factors whose acts are both autocrine and paracrine. Here, we used a model of surgical removal of POAT unilaterally and contralateral ovaries as controls, as both were under the same endocrine control, to assess the paracrine effect of the POAT on folliculogenesis. Surgical removal of unilateral POAT resulted in delayed antral follicular development and the increased number of atretic follicles, accompanied by decreased levels of intraovarian adipokines and growth factors, lipid accumulation and steroidogenic enzyme expression. POAT-deficient ovaries displayed compensatory increased expressions of intraovarian genes, such as Vegf and Adpn for angiogenesis, Acc, Fasn, and Gapdh involved in lipogenesis and Fshr in response to FSH stimulation. Furthermore, we demonstrated that removal of POAT promoted follicular apoptosis, caused retention of cytoplasmic YAP and inhibited PTEN-AKT-mTOR activation. These alterations were observed only in the POAT-deficient ovaries but not in the contralateral ovaries (with POAT), which suggests that a paracrine interaction between POAT and ovaries is important for normal folliculogenesis.

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Li-Jun Huo State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Cheng-Guang Liang State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Ling-Zhu Yu State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Zhi-Sheng Zhong State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Zeng-Ming Yang State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Heng-Yu Fan State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Da-Yuan Chen State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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Qing-Yuan Sun State Key Laboratory of Reproductive Biology, Institute of Zoology and Graduate School, Chinese Academy of Sciences, Beijing 100080, China, College of Life Science, Northeast Agricultural University, Harbin 150030, China and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

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The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In germinal vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The germinal vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after germinal vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I–telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in germinal vesicle breakdown and the anaphase–telophase transition.

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Wei Wang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Xia Chen College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Xinxiu Li College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Li Wang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Haiyan Zhang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Yu He College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Jingjing Wang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Yongyan Zhao College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Baole Zhang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Yinxue Xu College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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FSH plays a critical role in granulosa cell (GC) proliferation and steroidogenesis through modulation by factors including bone morphogenetic proteins family, which belongs to transforming growth factor β (TGFB) superfamily. TGFBs are the key factors in maintaining cell growth and differentiation in ovaries. However, the interaction of FSH and TGFB on the GCs' proliferation and steroidogenesis remains to be elucidated. In this study, we have investigated the role of SMAD4, a core molecule mediating the intracellular TGFB/SMAD signal transduction pathway, in FSH-mediated proliferation and steroidogenesis of porcine GCs. In this study, SMAD4 was knocked down using interference RNA in porcine GCs. Our results showed that SMAD4-siRNA causes specific inhibition of SMAD4 mRNA and protein expression after transfection. Knockdown of SMAD4 significantly inhibited FSH-induced porcine GC proliferation and estradiol production and changed the expression of cyclin D2, CDK2, CDK4, CYP19a1, and CYP11a1. Thus, these observations establish an important role of SMAD4 in the regulation of the response of porcine GCs to FSH.

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Chi-Huang Chen Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC
Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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Yu-Chi Yeh Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC
Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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Gwo-Jang Wu Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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Yen-Hua Huang Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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Wen-Fu Thomas Lai Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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Jah-Yao Liu Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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Chii-Ruey Tzeng Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC
Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC
Graduate Institute of Clinical Medicine, Departments of Obstetrics and Gynecology, Department of Psychiatry, School of Medicine, Department of Biochemistry and Graduate Institute of Medical Sciences, Center for Reproductive Medicine and Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC

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The applications of in vivo bioluminescent imaging (BLI) with a luciferase reporter gene occur widely across biomedical fields. Luciferase-transgenic mice are highly useful donors for tracking transplanted ovarian tissues. Realizing the full potential of this system may greatly benefit the study of the physiological behaviour and function of transplanted grafts, and the rapid and reliable evaluation of new transplantation protocols. The ovarian tissues of donor FVB/N-Tg(PolII–Luc)Ltc transgenic mice, with a luciferase transgene as the reporter, were transplanted into iso/allogeneic recipients. Rejection, ovarian function and BLI were quantitatively analysed in vivo over time. The BLI of the ovarian isografts revealed longer survival than that of allografts, even with cyclosporine A (CsA) treatment. The CD4+/CD8+ ratios of peripheral T-cells were significantly reduced in allografts compared with those in isografts (P<0.0001) during rejection, whereas CD19+ cell numbers were higher in allografts. The infiltration of CD4+/CD8+ cells into the graft was unremarkable in isografts from day 1, but was strong in allografts from day 8 onwards. Hormone activity revealed complete oestrus cycles in the isografts but only the dioestrus stage in the allografts. These results demonstrate that BLI in vivo expedites the fast throughput and fate maps of ovarian grafts. The use of BLI to longitudinally monitor ovarian grafts for immunorejection demonstrated the short survival of allografts and the much longer survival of isografts. CsA treatment alone is ineffective against the acute rejection of ovarian allografts.

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Zhen-Yu Zheng State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, College of Life Sciences, Northeast Agricultural University, Harbin 150030, People’s Republic of China and Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211, USA

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Qing-Zhang Li State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, College of Life Sciences, Northeast Agricultural University, Harbin 150030, People’s Republic of China and Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211, USA

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Da-Yuan Chen State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, College of Life Sciences, Northeast Agricultural University, Harbin 150030, People’s Republic of China and Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211, USA

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Heide Schatten State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, College of Life Sciences, Northeast Agricultural University, Harbin 150030, People’s Republic of China and Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211, USA

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Qing-Yuan Sun State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, College of Life Sciences, Northeast Agricultural University, Harbin 150030, People’s Republic of China and Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211, USA

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The protein kinase Cs (PKCs) are a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The phosphorylation of PKC in germ cells is not well defined. In this study, we described the subcellular localization of phopho-PKC in the process of mouse oocyte maturation, fertilization, and early embryonic mitosis. Confocal microscopy revealed that phospho-PKC (pan) was distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, phospho-PKC was localized in the vicinity of the condensed chromosomes, distributed in the whole meiotic spindle, and concentrated at the spindle poles. After metaphase I, phospho-PKC was translocated gradually to the spindle mid-zone during emission of the first polar body. After sperm penetration and electrical activation, the distribution of phospho-PKC was moved from the spindle poles to the spindle mid-zone. After the extrusion of the second polar body (PB2) phospho-PKC was localized in the area between the oocyte and the PB2. In fertilized eggs, phospho-PKC was concentrated in the pronuclei except for the nucleolus. Phospho-PKC was dispersed after pronuclear envelope breakdown, but distributed on the entire spindle at mitotic metaphase. The results suggest that PKC activation may play important roles in regulating spindle organization and stabilization, polar-body extrusion, and nuclear activity during mouse oocyte meiosis, fertilization, and early embryonic mitosis.

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Xiao-Wei Wei The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Shanghai Municipal Key Clinical Specialty, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Xue-Qing Liu The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Shanghai Municipal Key Clinical Specialty, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yu-Chen Zhang Department of Obstetrics and Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University of Medicine, Shanghai, China

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Chuan-Mei Qin The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Shanghai Municipal Key Clinical Specialty, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yi Lin The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Shanghai Municipal Key Clinical Specialty, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Fu-Ju Tian The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China
Shanghai Municipal Key Clinical Specialty, Shanghai, China
Institute of Birth Defects and Rare Diseases, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Recurrent pregnancy loss (RPL) is a multifactorial condition with no explanation of miscarriage in approximately half of the RPL patients, consequently leaving deep physical and emotional sequels. Transcription factor 3 (TCF3 or E2A), is a unique member of the LEF/TCF family and plays an important role in embryogenesis. However, its function in RPL is poorly understood. Using real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry, we demonstrated that TCF3 was downregulated in decidual tissues from RPL patients compared with healthy control (HC). Further, TCF3 knockdown inhibited proliferation, induced G0/G1 phase arrest, and promoted migration in human endometrial stromal cells (HESCs), while overexpression of TCF3 exhibited the opposite effects. RNA-sequencing analysis combined with gene-set enrichment analysis results showed that the mitogen-activated protein kinase pathway is potentially downstream of TCF3. Knockdown of TCF3 confirmed increased p38 phosphorylation, while overexpression of TCF3 inhibited p38 phosphorylation. Furthermore, we found that TCF3 protein level was decreased in HESCs under hypoxic incubation, while hypoxia-inducible factor-1α (HIF1A) knockdown increased the expression of TCF3. TCF3 overexpression recovered the proliferation ability of HESCs inhibited by hypoxia and reversed hypoxia-induced migration. Consistently, we found that RPL patients had a significantly higher level of HIF1A in the decidual tissue than HC. Overall, this study clarifies that increased HIF1A in the decidua contributes to the occurrence of RPL through the TCF3/p38 signaling pathway.

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